Preparation method of chitosan derivative and application of chitosan derivative to food preservation

A chitosan derivative and chitosan technology are applied in applications, dairy products, edible oil/fat, etc., to achieve good antibacterial and anti-oxidation preservation effects, good thermal stability, and to compensate for the effect of easy deactivation

Inactive Publication Date: 2018-01-16
TAIXING DONGSHENG FOOD TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the application of thermally stable recombinant bacterial laccases in the preparation of chitosan derivatives.

Method used

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  • Preparation method of chitosan derivative and application of chitosan derivative to food preservation
  • Preparation method of chitosan derivative and application of chitosan derivative to food preservation
  • Preparation method of chitosan derivative and application of chitosan derivative to food preservation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: Cloning of Bacillus vallismortis fmb-103 laccase gene

[0039] Bacillus vallismortis fmb-103 cells (CGMCCNo.6198) were collected by centrifugation, and the genomic DNA of Bacillus vallismortis fmb-103 was extracted with Shanghai Sangon Genomic DNA Extraction Kit.

[0040] Design two primers according to the registered Bacillus laccase gene (No.GU972592.1) in the Genebank database:

[0041] Upstream primer F-1: 5'-ATGACACTTGAAAAATTTGTGGATGC-3' (SEQ ID NO.3);

[0042] Downstream primer R-1: 5'-TTATTTATGGGGATCAGTTATATC-3' (SEQ ID NO.4);

[0043]In a 50 μl system, the final concentration of each primer is 1 μM, the final concentration of dNTPs is 0.2 mM, fmb-103 strain genomic DNA 10 ng, 2 U Pfu DNA polymerase. The amplification program was 94°C for 3min; 30×(94°C for 40s, 53°C for 50s, 72°C for 90s); 72°C for 10min. Agarose gel electrophoresis, gel cutting, recovery by Shanghai Sangon kit, connection of the recovered PCR product with TaKaRapMD19-simple-T v...

Embodiment 2

[0044] Embodiment 2: the construction of prokaryotic expression vector of Bacillus vallismortis fmb-103 laccase gene

[0045] According to the obtained laccase gene sequence, two primers were designed, the upstream primer plus the SacI recognition sequence, and the downstream primer plus the XhoI recognition sequence (the underlined part is the restriction enzyme recognition sequence):

[0046] Upstream primer F-2: 5′-CGC GAGCTC ATGACACTTGAAAAATTTGTGGATGC-3' (SEQ ID NO.5),

[0047] Downstream primer R-2: 5′-CCG CTCGAG TTATTTATGGGGATCAGTTATATC-3' (SEQ ID NO.6),

[0048] Add each component according to the following PCR system to amplify the laccase gene:

[0049]

[0050] The PCR program is: 94°C for 3min; 30×(94°C for 40s; 53°C for 50s; 72°C for 90s); 72°C for 10min.

[0051] Purify the PCR product with Shanghai Sangon PCR Product Purification Kit, add SacI, XhoI double enzyme digestion, inactivation, ethanol precipitation, ddH 2 O was redissolved, ligated with an app...

Embodiment 3

[0052] Embodiment 3: the expression of Bacillus vallismortis (Bacillus vallismortis) fmb-103 laccase gene in Escherichia coli

[0053] Transform the expression plasmid pET-23a-fmb-L103 containing fmb-L103 into Escherichia coli expression host strain BL21(DE3)pLysS, culture at 37°C for 10-11 hours, pick small colonies, insert into 50ml LB liquid culture containing ampicillin Base, 70-90rpm, 30°C culture overnight, according to the volume ratio of 1:40, take the seed solution and add it to 100ml LB liquid medium containing ampicillin, shake at 180rpm at 35°C for 2-3 hours until the OD600 is about 0.6, add IPTG (final Concentration 100μg / ml) induced. After 1.5 hours, the cells were collected by centrifugation. The bacterium was disrupted, and the supernatant was collected by centrifugation to obtain a crude enzyme solution of recombinant Bacillus vallismortis laccase (fmb-rL103).

[0054] The laccase activity was determined using the 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfon...

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Abstract

The invention discloses a preparation method of a chitosan derivative and application of the chitosan derivative to food preservation. According to the preparation method of the chitosan derivative, the chitosan derivative is obtained by performing reaction on chitosan powder in reaction liquid containing phenolic acid compounds and recombinant bacterial laccase at the temperature of 60 to 85 DEGC for 0.5 to 4 hours; and the phenolic acid compounds are selected from any one of ferulic acid, caffeic acid, chlorogenic acid, trihydroxybenzoic acid, gallic acid, catechinic acid, tannic acid and tea polyphenol. Compared with the chemical method, the preparation method of the chitosan derivative under catalyzed synthesis of bacterial laccase has the advantage of environmental friendliness and is a green production mode. The recombinant bacterial laccase has high heat stability, and catalytic reaction liquid can be reused repeatedly. The chitosan derivative has a good preservation effect invarious foods of fruit and vegetables, meat products, dairy products, grease products and the like.

Description

technical field [0001] The invention belongs to the field of food industry biotechnology, and relates to a preparation method of chitosan derivatives and its application in food preservation. Background technique [0002] Chitosan (CTS) is a derivative obtained by deacetylation of chitin under concentrated alkali conditions or in the presence of deacetylase. It is insoluble in water but soluble in Acidic natural polysaccharides are usually dissolved with organic acids (such as acetic acid) in research. Chitosan is safe, non-toxic and biodegradable. At the same time, as a natural renewable resource, it has high hydrophobicity, mechanical properties and antibacterial properties. It is widely used in food care and packaging, agriculture, biomedicine and sewage treatment. Wide range of applications. [0003] There are multiple active sites on the basic structural unit of chitosan, active hydroxyl groups exist on the C-3 and C-6 positions, and active amino groups (-NH 2 ), whe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04A23B7/08A23B4/20A23C3/08A23D9/06C11B5/00
Inventor 张充步国建张晓健孙建娜
Owner TAIXING DONGSHENG FOOD TECH
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